The elucidation of molecular alterations that occur during human breast cancer development may permit the identification of preventative strategies for women at high risk. Lesions such as atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) confer an increased risk for eventual patient development of invasive cancer. We have used in situ hybridization on a cohort of approximately 100 biopsy lesions to identify alterations in gene expression in early breast neoplastic progression. Initial data were obtained with probes encoding cell cycle proteins, cyclin D1 which is operative in G1, and cyclin A which is operative at the G2-M checkpoint. Quantitatively increased cyclin D1 mRNA expression was observed in only 18% of benign lesions, which confer no or only a slight increase in breast cancer risk, and 18% of premalignant ADH lesions, as compared to the matched normal ductal lobular units in the margins of each biopsy specimen. The transition to carcinoma was accompanied by frequent cyclin D1 mRNA overexpression, in 76% of low grade noncomedo DCIS, 87% of higher grade comedo DCIS and 83% of invasive breast carcinomas in the cohort. A similar trend was not observed using a riboprobe for cyclin A. The data identify cyclin D1 mRNA overexpression as the first known molecular event which may separate benign and premalignant human breast lesions from any form of breast carcinoma. Transfections of cyclin D1 cDNA, on both constitutive and inducible promoters, are underway in "normal" breast cultures, to determine if its overexpression causes or enhances early neoplastic progression. Similar in situ hybridization experiments are being conducted using the retinoic acid receptor family of genes. Data to date indicate that the RXR, but not RARalpha, beta, or gamma members of the family may be overexpressed in DCIS, but not in benign lesions. Additional cohort hybridizations and transfections are planned to further test the relevance of RXR mRNA expression to early breast neoplastic development.